Bone marrow (BM) is a soft spongy tissue that resides within the hollow cavity of long bones and represents about 4% of the total body weight. It is within the BM that blood cells are produced from pluripotent hematopoietic stem cells (HSC), a process referred to as hematopoiesis. It is also within the BM and lining the wall of compact bone (CB) that mesenchymal stem cells (MSC) reside. MSC are responsible for generating various cells of the body including fibroblasts, osteoblasts, chondrocytes, adipocytes, myocytes, and endothelial cells. In addition to HSC, hematopoietic monocytes, and MSC, there are several other types of cells that reside within the BM and CB making these tissues highly heterogeneous in nature.
Bone marrow provides specific microenvironments, or niches, necessary for hematopoietic and mesenchymal stem cells, osteoblasts, endothelial and other cells to co-exist and function. It is within these niches that decisions for a stem cell to become quiescent, proliferate, differentiate, and respond to external signals take place. Recently several reports have shown that cross-talk between different stem cell niches occur to elicit a proper response in vivo (7, 8, 9, 10). It is, therefore, not surprising that BM-derived cells as well as cells lining the inner walls of CB co-exist and affect each other in in vitro culture systems. The large number of cell types and niches present in the BM and CB and the inter-association that occurs among them often translates into heterogeneous in vitro cultures that contain several successful cell types that co-exist quite well. One recurring issue with mesenchymal stem cell (MSC) cultures from BM or crushed CB is the large number of unwanted contaminating macrophages, hematopoietic and possibly other non-mesenchymal cell populations that tend to overgrow the more rare population of MSC in culture. Cultures of BM and CB-derived MSCs are often overgrown with macrophages, which frequently reside on top of the growing MSC colonies, thereby interfering with MSC proliferation and behavior in culture. This in vitro heterogeneity is often unwanted in MSC research as experimental assays are designed to study or make use of that particular cell type without the interference of others.
CSF-1 is a cytokine that oligomerizes to CSF1R leading to trans-phosphorylation of this receptor to promote cell survival, proliferation, and differentiation of mononuclear phagocyte lineages into macrophages (4, 6). Consistent with its role in regulation of macrophage lineages, exogenous CSF-1 leads to increased production of monocytes and macrophages in mice (11), while non-functional CSF-1 (12) or CSF1R (13) mice display deficient numbers of macrophages resulting in diminished inflammatory response.
One of the phenotypic characteristics of hematopoietic monocytes is the expression of CSF1R on their cell surface membrane. On these cells, activation of CSF1R leads to proliferation and differentiation. Activation of this receptor is mediated via binding of the CSF-1 ligand to the CSF1R. This oligomerization elicits an adenosine triphosphate (ATP)-dependent tyrosine kinase-mediated transduction signal that ultimately directs hematopoietic monocytes to proliferate and/or differentiate.
GW2580 [5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl) pyrimidine-2,4-diamine]and KI20227 {N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N0-[1-(1,3-thiazole-2-yl)ethyl]urea} belong to a class of small molecules that specifically inhibit CSF1R kinase activity by competing with ATP binding to CSF1R kinase (14). It has been shown that GW2580 completely inhibits human CSF1R kinase at 60 nM while remaining inactive against 26 other kinases tested (3). Additionally, at 700 nM, GW2580 completely inhibited CSF-1-dependent growth of mouse myeloid cells, while a CSF-1-independent cell line, human fibroblasts, and other endothelial cells remained highly resistant to GW2580 (3).